Impacts of Dystrophin and Utrophin Domains on Actin Structural Dynamics: Implications for Therapeutic Design
- 作者：Ava Yun Lin ; Ewa Prochniewicz ; Davin M. Henderson ; Bin Li ; James M. Ervasti ; David D. Thomas ; ddt@umn.edu
- 关键词：TPA ; time-resolved phosphorescence anisotropy ; STR ; spectrin-type repeat ; wt ; wild type ; ErIA ; erythrosine iodoacetamide ; NIH ; National Institutes of Health
- 刊名：Journal of Molecular Biology
- 出版年：2012
- 期刊代码：102_00222836
- 类别：imm
- 出版时间：29 June, 2012
- 卷：420
- 期：1-2
- 页码：87-98
- 文件大小：1363 K
摘要
We have used time-resolved phosphorescence anisotropy (TPA) of actin to evaluate domains of dystrophin and utrophin, with implications for gene therapy in muscular dystrophy. Dystrophin and its homolog utrophin bind to cytoskeletal actin to form mechanical linkages that prevent muscular damage. Because these proteins are too large for most gene therapy vectors, much effort is currently devoted to smaller constructs. We previously used TPA to show that both dystrophin and utrophin have a paradoxical effect on actin rotational dynamics鈥攔estricting amplitude while increasing rate, thus increasing resilience, with utrophin more effective than dystrophin. Here, we have evaluated individual domains of these proteins. We found that a 鈥渕ini-dystrophin,鈥?lacking one of the two actin-binding domains, is less effective than dystrophin in regulating actin dynamics, correlating with its moderate effectiveness in rescuing the dystrophic phenotype in mice. In contrast, we found that a 鈥渕icro-utrophin,鈥?with more extensive internal deletions, is as effective as full-length dystrophin in the regulation of actin dynamics. Each of utrophin's actin-binding domains promotes resilience in actin, while dystrophin constructs require the presence of both actin-binding domains and the C-terminal domain for full function. This work supports the use of a utrophin template for gene or protein therapy designs. Resilience of the actin-protein complex, measured by TPA, correlates remarkably well with previous reports of functional rescue by dystrophin and utrophin constructs in mdx mice. We propose the use of TPA as an in vitro method to aid in the design and testing of emerging gene therapy constructs.